A focused deep mutational scan on the Env transmembrane and proximal cytosolic regions reveals an absence of sequence features for MA association
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PROVIDER: PRJNA521104 | ENA |
REPOSITORIES: ENA
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Project description:The HIV-1 surface glycoprotein Env binds target receptors to mediate fusion of the viral and host cell membranes during infection. Env is incorporated with very low density into virions, and in some cell types, regions within the long cytosolic C-terminal tail may mediate direct or indirect associations with Gag during virus assembly and budding. Here, the mutational landscape is determined across the transmembrane and proximal cytosolic domains of Env (001428 isolate from clade C) interacting with the MA domain of Gag at cellular membranes. No evidence is found in a derivative line of HEK293 cells for specific motifs that mediate Env/MA associations.
2019-02-07 | GSE126136 | GEO
Project description:This project seeks to identify protein-protein interactions with human endogenous retrovirus K envelope protein. A flag-tagged HERV-K env construct (fHERV-K env) was expressed in HEK293 cells. An anti-flag MAb was immobilized on magnetic protein G beads was used to capture fHERV-K env and interactors.
2020-07-06 | PXD019702 | Pride
Project description:The HIV-1 genome gains access to the inside of a cell via the mechanism of the viral spike protein Env, which undergoes a series of major conformational rearrangements after binding target receptors that ultimately drive virus-cell membrane fusion. Env is expressed as a heterogenous ensemble of conformations, which can inappropriately misdirect the host immune response towards the production of non-protective, strain-specific antibodies. Potent, broadly neutralizing antibodies (bnAbs) frequently recognize a ‘closed’ Env conformation, and therefore Env has undergone significant engineering to stabilize the closed state for vaccine incorporation. Previously, we used deep mutational scanning of Env from a prototypical tier 1 clade B strain (BaL) to characterize the sequence-activity landscape for binding to PG16, a bnAb that preferentially binds the closed state. Mutations were identified that increased expression of closed Env and reduced conformational heterogeneity, but these mutations were only partially transferable to Env sequences from other strains. To generate an expanded set of mutations that may be broadly applicable to diverse HIV-1 strains, we present here the deep mutational scanning of Env from the tier 2 clade C strain DU422 for interactions with CD4 and PG16. Residues across the trimerization domain and trimer interface have low mutational tolerance for maintaining PG16 recognition. New mutations are identified that enhance presentation of the closed Env conformation, and these are applied to Env sequences spanning multiple clades and tiers.
2019-02-01 | GSE117328 | GEO
Project description:HIV-1 infection begins with binding of the viral envelope glycoprotein Env to the host receptor CD4, triggering a series of conformational changes that lead to fusion of the virus and cell membranes. Env, a trimer of gp120 and gp41 subunits, occupies a ‘closed’ conformation with contacts between gp120 subunits at the apex, and transitions through an ‘open’ conformation with the gp120 subunits spread apart following CD4 binding. Using deep mutational scanning, sequence-fitness landscapes were mapped for full-length Env from the clade B BaL strain interacting with CD4, and broadly neutralizing antibodies VRC01 and PG16, which preferentially bind closed Env. Contacting residues are conserved for CD4 binding, and glycosylation at N262 is critical for accessing the high-affinity CD4-bound state. By comparison, VRC01 binding is resistant to most single amino acid substitutions, an ideal quality in a broadly neutralizing antibody. Also in contrast to CD4 interaction, Env interfacial residues are under tight selection for PG16 binding to maintain a closed conformation. Screening for mutations that enhanced PG16 binding, we identified several important sites, in particular neutralization of the electropositive apical cavity that we hypothesize promotes trimer opening by electrostatic repulsion. Mutations were combined to generate Quaternary Epitope Stabilized (QES) mutants with enhanced presentation of the PG16 epitope, and the mutations were partially transferable to other HIV-1 strains. These mutational analyses offer insight into Env conformational stabilization that may assist immunogen design.
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Project description:N-terminal acetylation is a major and vital protein modification catalysed by N-terminal acetyltransferases (NATs). NatF or Nα-acetyltransferase 60 (Naa60) was recently identified as a NAT in higher eukaryotes. Here, we found that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established Naa60 to face the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60 knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins, and that is has a preference for N-termini facing the cytosol. Moreover, NAA60 knockdown caused Golgi fragmentation, indicating an important role in maintenance of Golgi structural integrity. This work uncovers the first NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus far poorly characterized part of the N-terminal acetylome.
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Project description:N-glycans, which represent >50% mass of the HIV-1 envelope (Env) trimer, play important roles for virus-cell entry and immune evasion. How each glycan unit interacts to shape the Env protein-sugar complex and affects Env function is not well understood. Here, high-resolution glycomics analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation-site not only affected the Env structure at distant locations, but also had a ripple effect on Env-wide glycan processing, virus infectivity, and antibody recognition and virus neutralization. Specifically, the N262 glycan, although not located in the CD4-binding site, controlled Env binding to the CD4 receptor, affected the recognition of Env by several glycan-dependent broadly neutralizing antibodies, and altered heterogeneity of glycosylation at several sites, with N156, N160, and N448 displaying limited glycan processing. Molecular dynamic simulations visualized how specific oligosaccharide positions can move to compensate for loss of a glycan. This study demonstrates how changes in individual glycan units can alter molecular dynamics and processing of the Env-glycan shield and, consequently, Env function.
2021-09-09 | PXD017941 | Pride
Project description:CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Therefore, gaining insights into Tfh subsets promoting antibody persistence and functional capacity is crucial for vaccine design. We investigated the potential of inducing a mixed Tfh1/17 response to enhance HIV-1 Envelope (Env) antibodies to a DNA-prime/protein boost platform, utilizing rhesus macaques as our experimental model. Following immunization with Clade C gp140 protein formulated with cationic liposome-based formulation (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells, in contrast to the predominance of GC Tfh1 cells induced with gp140 formulated in monophosphoryl lipid A+QS-21 (MPLA). Analysis of lymph nodes, employing proteomic and transcriptional approaches, demonstrated robust induction of GC responses across vaccine platforms with distinct qualitative and quantitative effects elicited by MPLA versus CAF01. While the induction of GC Tfh1 cells with MPLA and GC Tfh1/17 cells with CAF01 resulted in comparable peak HIV-Env antibody levels, there was a notable difference in antibody persistence. The MPLA group demonstrated significantly greater antibody persistence at week 8 and up to 30 weeks after final immunization compared to CAF01. Inducing GC Tfh1 cells with MPLA furthermore enhanced tier 1 neutralization titers, antibody functions, and Env-specific IgG in the rectal mucosa. Notably, IFNγ+ Env-specific Tfh responses, both in blood and lymph nodes, were higher with MPLA and correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction may confer protective immunity against HIV.
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Project description:VRC01-class broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) and are of major interest for vaccine design. Unlike mature antibodies, corresponding VRC01-class germline precursors poorly bind to Env due to their weak ability to overcome the steric hindrance imparted by the glycans surrounding the CD4 BS. To date, immunogen design initiatives have largely relied on removal of these glycans and have met limited success in eliciting VRC01-class bnAbs. To understand elicitation of such bnAbs in humans, we structurally characterized the inferred germline precursor of VRC01 in complex with wild-type core gp120 by X-ray crystallography and modified trimeric 426c Env constructs by single-particle electron microscopy. We then validated glycan length and composition of germline VRC01-compatible Env constructs by EThcD LC-MS/MS. Our results reveal that engagement of the VRC01 germline antibody by a wild-type 426c gp120 is possible and can be enhanced by tailoring glycan length in the vicinity of the CD4BS.
2018-11-21 | PXD011494 | Pride
Project description:The high mutation rate of HIV is linked to the generation of viruses expressing proteins with altered function whose impact on disease progression is unknown. We investigated the effects of HIV-1 viruses lacking Env, Vpr and Nef on CD4+ T cell gene expression using high-density DNA microarray analysis and functional assays. Experiment Overall Design: Human activated CD4+ T-lymphocytes from three independent donors were infected with HIV-1 viruses that lack Env and Nef (pNL4-3.eGFP.R+E- or HIVD2GFP) or Env, Vpr and Nef. (pNL4-3.eGFP.R-E- or HIVD3GFP) were pseudotyped with VSVG envelope. As a control, CD4+ T-lymphocytes were infected with VSVG-pseudotyped eGFP. CD4+ T-cells were sorted 48 hours after infection using GFP as a marker of infectivity. RNA was isolated 10 hours after sorting, labeled, and prepared for microarray analysis.
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